An in vitro model of the tumor-lymphatic microenvironment with simultaneous transendothelial and luminal flows reveals mechanisms of flow enhanced invasion.

The most common cancers, including breast and skin, disseminate initially through the lymphatic system, yet the mechanisms by which tumor cells home towards, enter and interact with the lymphatic endothelium remain poorly understood. Transmural and luminal flows are important biophysical cues of the lymphatic microenvironment that can affect adhesion molecules, growth factors and chemokine expression as well as matrix remodeling, among others. Although microfluidic models are suitable for in vitro reconstruction of highly complex biological systems, the difficult assembly and operation of these systems often only allows a limited throughput. Here we present and characterize a novel flow chamber which recapitulates the lymphatic capillary microenvironment by coupling a standard Boyden chamber setup with a micro-channel and a controlled fluidic environment. The inclusion of luminal and transmural flow renders the model more biologically relevant, combining standard 3D culture techniques with advanced control of mechanical forces that are naturally present within the lymphatic microenvironment. The system can be monitored in real-time, allowing continuous quantification of different parameters of interest, such as cell intravasation and detachment from the endothelium, under varied biomechanical conditions. Moreover, the easy setup permits a medium-high throughput, thereby enabling downstream quantitative analyses. Using this model, we examined the kinetics of tumor cell (MDA-MB-231) invasion and transmigration dynamics across lymphatic endothelium under varying flow conditions. We found that luminal flow indirectly upregulates tumor cell transmigration rate via its effect on lymphatic endothelial cells. Moreover, we showed that the addition of transmural flow further increases intravasation, suggesting that distinct flow-mediated mechanisms regulate tumor cell invasion.

Design, fabrication, and characterization of a composite scaffold for bone tissue engineering.

Poly(lactide-co-glycolide) (PLGA) scaffolds have been successfully used in bone tissue engineering, with or without hydroxyapatite (HA) and with a macroporosity given either by simple PLGA sphere packaging and/or by leaching out NaCl. The objective of this work was the optimization of the design parameters for bone tissue engineering scaffolds made by sintering microspheres of PLGA, HA nanocrystals for matrix reinforcement and osteoconduction, and salt crystals for macroporosity and control of matrix pore size. Microsphere fabrication by a single-emulsion and solvent evaporation technique was first optimized to obtain a high yield of PLGA microspheres with a diameter between 80 and 300 microm. The influence of the sintering process and matrix composition on the scaffold structure was then evaluated morphologically and mechanically. Three scaffold types were tested for biocompatibility by culturing with human fibroblasts for up to 14 days. The most important parameters to obtain microspheres with the selected diameter range were the viscosity ratio of the dispersed phase to the continuous phase and the relative volume fraction of the 2 phases. The Young's modulus and the ultimate strength of the sintered matrices ranged between 168-265 MPa and 6-17 MPa, respectively, within the range for trabecular bone. Biocompatibility was demonstrated by fibroblast adhesion, proliferation, and spreading throughout the matrix. This work builds upon previous work of the PLGA/HA sintering technique to give design criteria for fabricating a bone tissue engineered matrix with optimized morphological, functional, and biological properties to fit the requirements of bone replacements.

Lymphatic function, lymphangiogenesis, and cancer metastasis.

The lymphatic system serves as the primary route for the metastasis of many cancers and the extent of lymph node involvement is the most important indicator of tumor aggressiveness. Despite the apparent importance of the lymphatic vessels for tumor dissemination, it has remained unclear whether activation of lymphatic endothelial cells may affect tumor progression and metastasis and the molecular mechanisms of lymphangiogenesis are just beginning to be elucidated. This overview describes the unique structural and functional characteristics of the lymphatic vessels that render them particularly suitable for invasion by tumor cells and for their efficient transport to lymph nodes. Recent evidence indicates occurrence of tumor lymphangiogenesis and its correlation with metastasis. Molecular regulation of tumor lymphangiogenesis, its significance for tumor metastasis, and implications for cancer therapy are discussed.

The physiology of the lymphatic system.

This paper presents an overview of the anatomy, physiology, and biology of the lymphatic system specifically relevant to lymphatic drug delivery. We will briefly review the classic fluid and solute transport literature, and also examine the current research in lymphatic endothelial cell biology and tumor metastasis in the lymphatics because of the increasing potential for targeted delivery of immunomodulators, chemotherapeutics, and genetic material to specific lymph nodes (Refs. [1-7]).

Mechanical stress is communicated between different cell types to elicit matrix remodeling.

Tissue remodeling often reflects alterations in local mechanical conditions and manifests as an integrated response among the different cell types that share, and thus cooperatively manage, an extracellular matrix. Here we examine how two different cell types, one that undergoes the stress and the other that primarily remodels the matrix, might communicate a mechanical stress by using airway cells as a representative in vitro system. Normal stress is imposed on bronchial epithelial cells in the presence of unstimulated lung fibroblasts. We show that (i) mechanical stress can be communicated from stressed to unstressed cells to elicit a remodeling response, and (ii) the integrated response of two cell types to mechanical stress mimics key features of airway remodeling seen in asthma: namely, an increase in production of fibronectin, collagen types III and V, and matrix metalloproteinase type 9 (MMP-9) (relative to tissue inhibitor of metalloproteinase-1, TIMP-1). These observations provide a paradigm to use in understanding the management of mechanical forces on the tissue level.

Role of extracellular matrix assembly in interstitial transport in solid tumors.

The extracellular matrix (ECM) may contribute to the drug resistance of a solid tumor by preventing the penetration of therapeutic agents. We measured differences in interstitial resistance to macromolecule (IgG) motion in four tumor types and found an unexpected correspondence between transport resistance and the mechanical stiffness. The interstitial diffusion coefficient of IgG was measured in situ by fluorescence redistribution after photobleaching. Tissue elastic modulus and hydraulic conductivity were measured by confined compression of excised tissue. In apparent contradiction to an existing paradigm, these functional properties are correlated with total tissue content of collagen, not glycosaminoglycan. An extended collagen network was observed in the more penetration-resistant tumors. Collagenase treatment of the more penetration-resistant tumors significantly increased the IgG interstitial diffusion rate. We conclude that collagen influences the tissue resistance to macromolecule transport, possibly by binding and stabilizing the glycosaminoglycan component of the ECM. These findings suggest a new method to screen tumors for potential resistance to macromolecule-based therapy. Moreover, collagen and collagen-proteoglycan bonds are identified as potential targets of treatment to improve macromolecule delivery.

Cells shed from tumours show reduced clonogenicity, resistance to apoptosis, and in vivo tumorigenicity.

The goal of this study was to compare growth characteristics of cells shed from a tumour with the native tumour cells. The human colon adenocarcinoma LS174T and its highly metastatic subline LS LiM 6 were grown as tissue-isolated tumours in nude mice and perfused to collect shed cells. The tumours were then excised and prepared into single-cell suspensions. Clonogenicity in 0.3-0.9% agarose, apoptotic fraction, and in vivo tumorigenicity were determined for each population. In both tumour lines, shed cells were less clonogenic, more apoptotic and less tumorigenic than cells isolated directly from their native tissue. These findings suggest that shed cells have a low metastatic potential compared to native tumour cells, most likely because they represent an apoptotic population.

Mechanics of interstitial-lymphatic fluid transport: theoretical foundation and experimental validation.

Interstitial fluid movement is intrinsically linked to lymphatic drainage. However, their relationship is poorly understood, and associated pathologies are mostly untreatable. In this work we test the hypothesis that bulk tissue fluid movement can be evaluated in situ and described by a linear biphasic theory which integrates the regulatory function of the lymphatics with the mechanical stresses of the tissue. To accomplish this, we develop a novel experimental and theoretical model using the skin of the mouse tail. We then use the model to demonstrate how interstitial-lymphatic fluid movement depends on a balance between the elasticity, hydraulic conductivity, and lymphatic conductance as well as to demonstrate how chronic swelling (edema) alters the equipoise between tissue fluid balance parameters. Specifically, tissue fluid equilibrium is perturbed with a continuous interstitial infusion of saline into the tip of the tail. The resulting gradients in tissue stress are measured in terms of interstitial fluid pressure using a servo-null system. These measurements are then fit to the theory to provide in vivo estimates of the tissue hydraulic conductivity, elastic modulus, and overall resistance to lymphatic drainage. Additional experiments are performed on edematous tails to show that although chronic swelling causes an increase in the hydraulic conductivity, its greatly increased distensibility (due to matrix remodeling) dampens the driving forces for fluid movement and leads to fluid stagnation. This model is useful for examining potential treatments for edema and lymphatic disorders as well as substances which may alter tissue fluid balance and/or lymphatic drainage.

Return of lymphatic function after flap transfer for acute lymphedema.

OBJECTIVE: The goals of this work were to develop animal models of lymphedema and tissue flap transfer, and to observe physiologic changes in lymphatic function that occur in these models over time, both systemically with lymphoscintigraphy (LS) and locally using fluorescence microlymphangiography (FM). SUMMARY BACKGROUND DATA: Although lymphedema has been managed by a combination of medical and surgical approaches, no effective long-term cure exists. Surgical attempts aimed at reconnecting impaired lymphatic channels or bypassing obstructed areas have failed. METHODS: The tails of rats (A groups) and mice (B groups) were used because of their different features. Lymphedema was created by ligation of the lymphatics at the tail base and quantified by diameter measurements there. In the experimental group, rectus abdominis myocutaneous flap was transferred across the ligation. In addition to the ligation (A1 and B1) and ligation + flap (A2 and B2) groups, three control groups were included: sham flap with ligation (B4), sham flap alone (B5), and normal (A3 and B3) animals. Observations were made at weekly time points for lymphatic function and continuity. RESULTS: Lymphedema was successfully created in the mouse ligation groups (B1 and B4) and sustained for the entire length of observation (up to 14 weeks). Lymphatic continuity was restored in those animals with transferred flaps across the ligation site (A2 and B2), as seen both by LS and FM. Sham flaps did not visibly affect lymphatic function nor did they cause any visible swelling in the tail. CONCLUSIONS: Acute lymphedema developing after ligation of tail lymphatics in mice can be prevented by myocutaneous flap transfer. Restored lymphatic continuity and function were demonstrable using lymphoscintigraphy and fluorescence microlymphangiography.

Influence of intravenous perfluorocarbon administration on the dynamic behavior of lung surfactant.

Intravenous administration of perfluorocarbon (PFC) compounds can lead to pulmonary hyperinflation and respiratory distress in some mammals. This study was designed to quantify the effects of two PFC emulsions on the dynamic behavior of lung surfactant and to demonstrate that PFC is retained in the liquid lining the lung. New Zealand White rabbits received isotonic saline (3 ml/kg), Fluosol (15 ml/kg) or Oxygent (90% perfluorooctyl-bromide emulsion, 3 ml/kg). After seven days we euthanized the animals and lavaged the lungs. Surface tension-surface area relationships (sigma-A loops) were measured with the lavage fluid placed in a Wilhelmy plate-oscillating bellows apparatus. Loop hysteresis area after Fluosol administration was 334 +/- 92 dyne-cm, significantly greater than after saline (203 +/- 36 dyne-cm) but not Oxygent (274 +/- 66 dyne-cm). Loop hysteresis slope was higher with Oxygent (0.8 +/- 0.4 dyne/cm3) than after saline (0.6 +/- 0.3 dyne/cm3) or Fluosol (0.5 +/- 0.1 dyne/cm3). 282 MHz 19F NMR spectral analysis demonstrates that both PFCs tested appear only in the extracellular fraction of the lavage fluid. These results show that pulmonary elimination of intravascular PFC leads to PFC presence in the liquid lining the airways where it alters surfactant dynamic mechanical behavior.

Perfluorocarbon induced alterations in pulmonary mechanics.

Perfluorocarbon (PFC) compounds induce pulmonary hyperinflation and respiratory distress in some animals following intravenous administration. This study was designed to quantify the effects of two PFC emulsions on lung volumes and compliance and to identify the mechanism of pulmonary hyperinflation. New Zealand White rabbits received isotonic saline (3 ml/kg), Fluosol (15 ml/kg) or Oxygent (90% perfluorooctyl-bromide emulsion, 3 ml/kg). After seven days we measured functional residual capacity, vital capacity, lung compliance and thoracic gas volume. Gross and microscopic histologic examination of the lungs was performed. Functional residual capacity after Fluosol administration was 16.0 +/- 4.0 ml/kg, significantly greater than after saline (3.4 +/- 1.0 ml/kg) or Oxygent (4.0 +/- 1.4 ml/kg). Vital capacity was lower with Fluosol (30 +/- 5.0 ml/kg) than after saline (37 +/- 3.0 ml/kg) or Oxygent (37 +/- 2.0 ml/kg). Thoracic gas volume increased from 9 +/- 1.0 ml/kg (saline) to 16 +/- 13 ml/kg (Oxygent) and 33 +/- 7.0 ml/kg (Fluosol). Lung compliance was the same after saline (1.6 +/- 0.5 ml.cm H2O-1.kg-1) and Oxygent (1.5 +/- 0.3 ml.cm H2O-1.kg-1) but lower after Fluosol (0.9 +/- 0.1 ml.cm H2O-1.kg-1). Gross pathology demonstrated foam exudation from airways of animals receiving PFCs and intra-alveolar foam was identified by light microscopy. These results show intra-airway foam formation causes gas trapping and shifts tidal breathing to a less compliant region of the pressure-volume curve.

Unilateral adrenal enlargement due to Histoplasma capsulatum.

Human infection with Histoplasma capsulatum runs the gamut from asymptomatic to disseminated disease. CT-directed fine-needle aspiration of bilaterally enlarged adrenal glands has been used in diagnosing serious infections with this ubiquitous organism. Three cases have previously been reported in which H. capsulatum infection caused unilateral adrenal enlargement; this enlargement was diagnosed post-mortem. We describe three patients with unilateral adrenal enlargement due to H. capsulatum whose conditions were diagnosed antemortem. We encourage clinicians to include infection with H. capsulatum as well as other granulomatous diseases and tumors in the differential diagnosis of unilateral adrenal enlargement.

Methods of documenting cost savings associated with an antibiotic management program.

Documenting the financial impact of an antibiotic management program is important for demonstrating the value of these pharmacy-driven or interdisciplinary services. There are several cost-saving methods that may be utilized in justifying such programs. These include intervention analysis, drug purchasing comparisons, specific agent tracking, patient length of stay analysis, and evaluation of antimicrobial therapy for a specific disease state. Several of these methods have been utilized for successfully documenting the financial impact generated by our institutions antibiotic management program.

Transport in lymphatic capillaries. I. Macroscopic measurements using residence time distribution theory.

We present a novel integrative method for characterizing transport in the lymphatic capillaries in the tail of the anesthetized mouse, which is both sensitive and reproducible for quantifying uptake and flow. Interstitially injected, fluorescently labeled macromolecules were used to visualize and quantify these processes. Residence time distribution (RTD) theory was employed to measure net flow velocity in the lymphatic network as well as to provide a relative measure of lymphatic uptake of macromolecules from the interstitium. The effects of particle size and injection pressure were determined. The uptake rate was found to be independent of particle size in the range of a 6- to 18-nm radius; beyond this size, the interstitial matrix seemed to pose a greater barrier. A comparison of 10 vs. 40 cmH2O injection pressure showed a significant influence on the relative uptake rate but not on the net velocity within the network (3.3 +/- 0.8 vs. 3.8 +/- 1.0 micron/s). This suggested the presence of a systemic driving force for baseline lymph propulsion that is independent of the local pressure gradients driving the uptake. This model can be used to examine various aspects of transport physiology of the initial lymphatics.

Transport in lymphatic capillaries. II. Microscopic velocity measurement with fluorescence photobleaching.

Despite its relevance to the physiology of lymph formation and propulsion, the instantaneous flow velocity in single lymphatic capillaries has not been measured to date. The method of fluorescence recovery after photobleaching (FRAP) was adapted for this purpose and used to characterize flow in the lymphatic capillaries in tail skin of anesthetized mice during a constant-pressure intradermal injection of fluorescein isothiocyanate-dextran (mol wt 2 x 10(6). The median lymph flow velocity was 4.7 microns/s, and the velocity magnitude ranged from 0 to 29 microns/s. The direction of flow was generally proximal, but stasis and backflow toward the site of injection was also detected. Evidence for oscillatory flow was detected in some FRAP experiments, and in separate experiments a periodicity of approximately 120 min-1, directly correlated to respiration frequency, was measured by tracking the motion of fluorescent latex microspheres (1 micron diam) introduced into the lymphatic capillary network. The velocity magnitude showed a correlation with duration of infusion but not with distance from injection site. It is speculated that the temporal decay of mean velocity magnitude could be related to the relaxation of local pressure gradients as partially collapsed vessels expand during the infusion.

Clinical experience with HIV-infected patients at the University of Oklahoma Health Sciences Center: an update.

We reviewed the course of 545 human immunodeficiency virus (HIV)-infected patients seen between 1983 and March 30, 1991. A majority were Caucasian homosexual or bisexual men, while parenteral drug abusers represented a smaller proportion than seen nationwide. In the 274 patients with the acquired immunodeficiency syndrome (AIDS), the distribution of AIDS-defining conditions was generally consistent with those reported in studies from elsewhere in the United States. However, toxoplasmosis remained relatively uncommon. There was a slightly higher incidence of disseminated histoplasmosis compared to other studies. HIV encephalopathy (AIDS dementia) was likely underdiagnosed. Although data suggested prolongation of the asymptomatic phase of HIV infection, median survival after AIDS diagnosis remained approximately 12 months.

Catalase-negative listeria monocytogenes causing meningitis in an adult. Clinical and laboratory features.

A 63-year-old previously healthy woman presented with acute meningitis. Cultures of the cerebrospinal fluid yielded a serotype 1/2a isolate of Listeria monocytogenes that was biochemically typical in all respects, other than the reproducible lack of catalase production. During therapy, the patient developed oculomotor dysfunction that was attributed to an abscess in the internal capsule. This case report documents the existence of catalase-negative L. monocytogenes indicating that catalase production should not be a strict criterion for identification of Listeria. Furthermore, this clinical experience extends in vitro and experimental animal studies indicating that catalase production is not a necessary virulence factor for invasion by Listeria.